1frn
From Proteopedia
(New page: 200px<br /><applet load="1frn" size="450" color="white" frame="true" align="right" spinBox="true" caption="1frn, resolution 2.0Å" /> '''THE INVOLVEMENT OF SE...) |
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| - | [[Image:1frn.gif|left|200px]]<br /><applet load="1frn" size=" | + | [[Image:1frn.gif|left|200px]]<br /><applet load="1frn" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1frn, resolution 2.0Å" /> | caption="1frn, resolution 2.0Å" /> | ||
'''THE INVOLVEMENT OF SER96 IN THE CATALYTIC MECHANISM OF FERREDOXIN-NADP+ REDUCTASE: STRUCTURE-FUNCTION RELATIONSHIP AS STUDIED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY'''<br /> | '''THE INVOLVEMENT OF SER96 IN THE CATALYTIC MECHANISM OF FERREDOXIN-NADP+ REDUCTASE: STRUCTURE-FUNCTION RELATIONSHIP AS STUDIED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that | + | The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS) |
==About this Structure== | ==About this Structure== | ||
| - | 1FRN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Spinacia_oleracea Spinacia oleracea] with PO4, SO4 and FAD as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ferredoxin--NADP(+)_reductase Ferredoxin--NADP(+) reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.1.2 1.18.1.2] Full crystallographic information is available from [http:// | + | 1FRN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Spinacia_oleracea Spinacia oleracea] with <scene name='pdbligand=PO4:'>PO4</scene>, <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=FAD:'>FAD</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ferredoxin--NADP(+)_reductase Ferredoxin--NADP(+) reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.18.1.2 1.18.1.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1FRN OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Spinacia oleracea]] | [[Category: Spinacia oleracea]] | ||
| - | [[Category: Bruns, C | + | [[Category: Bruns, C M.]] |
| - | [[Category: Karplus, P | + | [[Category: Karplus, P A.]] |
[[Category: FAD]] | [[Category: FAD]] | ||
[[Category: PO4]] | [[Category: PO4]] | ||
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[[Category: oxidoreductase (nadp+(a)]] | [[Category: oxidoreductase (nadp+(a)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:41:55 2008'' |
Revision as of 10:41, 21 February 2008
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THE INVOLVEMENT OF SER96 IN THE CATALYTIC MECHANISM OF FERREDOXIN-NADP+ REDUCTASE: STRUCTURE-FUNCTION RELATIONSHIP AS STUDIED BY SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY
Overview
The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
About this Structure
1FRN is a Single protein structure of sequence from Spinacia oleracea with , and as ligands. Active as Ferredoxin--NADP(+) reductase, with EC number 1.18.1.2 Full crystallographic information is available from OCA.
Reference
Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography., Aliverti A, Bruns CM, Pandini VE, Karplus PA, Vanoni MA, Curti B, Zanetti G, Biochemistry. 1995 Jul 4;34(26):8371-9. PMID:7677850
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